This invention relates to the identification of novel functional sites on the urokinase receptor in the presence of the receptor binding region of urokinase. Described herein are peptides derived from bacteriophage display that identify the sites, and a general method for identifying functional sites on proteins using bacteriophage display. Also, methods of using urokinase receptor functional sites for studies of vitronectin and integrin interaction with urokinase:urokinase receptor complex interaction are described. Also described are uses of the instant peptides for developing therapeutic molecules capable of antagonisting interactions of the vitronectin and integrin peptides with the urokinase:urokinase receptor complex.
The urokinase plasminogen activator (uPA) is a serine protease that interacts with its cell surface receptor (uPAR) providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. The uPA:uPAR complex converts plasminogen into plasmin which is known to degrade various matrix glycoproteins as described in Ellis et al, J. Biol. Chem. 264: 2185-2188 (1989), Vassili et al, J. Clin. Invest. 88: 1067-1072 (1991), and Mignatti and Rifkin, Physiol. Rev. 73: 161-195 (1993). The simaltaneous expression of uPA and its receptor has been associated with localized plasminogen activation and pericellular matrix degradation during directed cell migration of normal and tumor cells.
The urokinase receptor (uPAR) is a 283 amino acid glycosylphosphatidyl-inositol (GPI)-anchored receptor protein of urokinase and vitronectin which appears to be a triplication of a 90 amino acid domain as described in Plough, and Ellis, FEBS Lett. 349:163-168 (1994) and Roldan et al, EMBO J. 9: 467-474 (1990). Proteolysis of uPAR can yield fragments composed of domain 1 and domains 2-3, and subsequent analysis has shown that disulfide bonding pattern of domain 1 is completely internal to the domain, as described in Plough et al, J. Biol. Chem. 268:17539-17546 (1993). and Kieffer et al, Biochem. 33:4471-4482 (1994).
The migration and invasion of cells appear to require cell surface localized proteolysis and adhesion to specific components of the extracellular matrix. These processes are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis as described in Fazioli et al, Trends Pharmacol.Sci. 15:25-29(1994), and Mignatti et al, Physiol.Rev. 73:161-195 (1993). Important components of the cell surface proteolytic and cellular adhesion cascades are the plasminogen activator/plasmin system, matrix metalloproteinases, and integrins, as described in Felding-Habermann et al, Curr.Biol. 5 864-868 (1993). Adhesion to the extracellular matrix component vitronectin has been reported to correlate with UPAR expression, and uPA binding sites and vitronectin receptors have been shown to colocalize on HT1080 cells, as described in Waltz et al, J.Biol.Chem. 269: 14746-14750 (1994)., and Ciambrone et al, J.Biol.Chem. 267: 13617-13622 (1992). More recently it has been demonstrated that uPAR can function as a cell adhesion receptor for vitronectin in a uPA dependent manner as described in Wei et al, J.Biol.Chem. 269: 32380-32388 (1994).
Early experiments using chemical cross-linking suggested that the first domain of uPAR was sufficient for high affinity binding of uPA, however, subsequent work has shown that an intact 3-domain molecule is required, and that additional binding determinants in domains 2 and 3 are likely involved, as described in Plough et al, Biochem. 3: 8991-8997 (1994). The undefined interactions may be with the uPA EGF-like domain or indirect interactions affecting the conformation of domain 1. Previous work has been unsuccessful in distinguishing whether domain 2 and 3 has measurable affinity for uPA, because of the difficulty of separating domain 2 and 3 from trace amounts of full length uPAR as described in Plough et al, Biochem. 3: 8991-8997 (1994).
The uPA:uPAR system has been identified as promoting pericellular proteolysis, and functions attributable to uPAR include cell migration, adhesion and mitogenesis. It would be desirable, therefore, to elucidate the function of domains 2 and 3 of uPAR.
A first embodiment of the invention is a method of identifying an orphan binding site on a target polypeptide sequence by
(a) providing
(1) a library of potential ligands,
(2) a target polypeptide in contact with a known ligand for the target polypeptide,
(b) contacting the target polypeptide and known ligand with the library of potential ligands, and
(c) identifying the potential ligand that binds to the target polypeptide in the presence of the known ligand to form a binding pair with the target polypeptide and known ligand.
Another embodiment of the invention is an isolated peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to an integrin. The isolated peptide can be YHXLXXGYMYT (SEQU ID NO:5) or AESTYHHLSLGYMYTLN (SEQ ID NO:4).
Another embodiment of the invention is an isolated peptide that binds a urokinase plasminogen activator (uPAR) and inhibits uPAR binding to vitronectin. The isolated peptide can be AEPVYQYELDSYLRSYY (SEQ ID NO: 1), AEFFKLGPNGYVYLHSA (SEQ ID NO:2), or AELDLSTFYDIQYLLRT (SEQ ID NO:3) or FKLXXXGYVYL (SEQ ID NO:6).
Yet another embodiment of the invention is an isolated nucleic acid sequence that encodes a peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to an integrin. The isolated nucleic acid sequence can encode the amino acid sequence of YHXLXXGYMYT (SEQ ID NO:5) or STYHHLSLGYMYTLN (SEQ ID NO:4).
Still another embodiment of the invention is an isolated nucleic acid sequence that encodes a peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to vitronectin. The isolated nucleic acid sequence can encode the amino acid sequence of AEPVYQYELDSYLRSYY (SEQ ID NO: 1), or FFKLGPNGYVYLHSA (SEQ ID NO:2) or, AELDLSTFYDIQYLLRT (SEQ ID NO:3) or FKLXXXGYVYL (SEQ ID NO:6).
Yet another embodiment of the invention is a method of treating a patient with a disorder characterized by upregulation of uPA and uPAR by providing an effective amount of an antagonist of a uPAR:integrin binding pair, and administering the antagonist to the patient.
An additional embodiment of the invention is a method of screening for an antagonist of uPAR:integrin interaction comprising the steps of providing a peptide antagonist of a uPAR:integrin interaction, competing the peptide antagonist with a candidate antagonist for binding to uPAR, and identifying a candidate antagonist by the ability to compete with the peptide antagonist for uPAR binding.
Still a further embodiment of the invention is a small molecule antagonist of a uPAR:integrin interaction identified by the just described method; a peptide antagonist of a uPAR:integrin interaction identified by that method, and a peptoid antagonist of a uPAR:integrin interaction identified by the same method.
Another embodiment of the invention is a pharmaceutical composition for treating a disorder characterized by upregulation of uPA and uPAR comprising an effective amount of an antagonist of a uPAR:integrin binding pair and a pharmaceutically acceptable carrier.
Yet another embodiment of the invention is a pharmaceutical composition for treating a patient with a disorder characterized by upregulation of uPA and uPAR comprising an effective amount of a nucleic acid encoding a peptide antagonist of a uPAR:integrin binding pair and a pharmaceutically acceptable carrier suitable for expressing the peptide in the patient.